Biological Evaluation of the Isolated Compounds from Methanol Fraction of Leutea avicennia Mozaff

Leutea avicennia Mozaff. That belongs to Apiaceae family is an endemic species distributed in the west of Iran. The aim of this study was to investigate the antioxidant activity and acetylcholinestrase (AChE) inhibition of the crude extract, fractions, and isolated compounds from methanol fraction of L. avicenniae. Five compounds were detected from methanol fraction; three phenolic compounds as p-coumric acid, caffeic acid, salicylic acid and also, two flavonoids as quercetin and astragalin. These structures were identified by spectroscopic techniques such as 1H-NMR, 13C-NMR, and UV. Antioxidant activity was evaluated by the free radical scavenging assay using 2, 2-diphenyl-1-picryl- hydrazyl (DPPH) method. Ellman colorimetric method was used to determine acetylcholinestrase (AChE) inhibition. In the DPPH assay, Quercetin exerted the highest antioxidant activity (IC50 = 10.24 ± 1.3 µg/mL). Caffeic acid inhibited AChE with IC50 = 12.06 ± 2.01µg/mL which were comparable to Galanthamine as positive control (IC50 = 62.44 ± 2.2µg/mL). In conclusion, methanol extract of L. avicenniae contains bioactive components with antioxidant and AChE inhibitory effects.


Introduction
Free radicals, along with oxidative stress reactions can lead to damage biological molecules like DNA, proteins, and lipids (1, 2). Therefore, oxidative stress has been involved in pathogenesis of various diseases including cancers, atherosclerosis, and neurodegenerative disorders such as Alzheimer'disease (AD) (3,4). Pathophysiology of AD is relevant to oxidative stress and evacuation of neurotransmitter acetylcholine by acetylcholinesterases (5). A prevalent method for treating AD is to increase the ACh level in the brain utilization AChE inhibitors (6). The chemical medicines have limitations associated with side-effects. The search for discovering strong new AChE inhibitors from natural origins without side effects is great attention (7). Currently, a number of treatments are applied against Alzheimer's disease too to answer the effect of oxidative stress. These consist of the use of acetylcholinesterase inhibitors (AChEIs) and large amount of antioxidant (5).
Plants supply a valuable resource, functional as leads to the extension of therapeutic compounds (8). Regular consumption of numerous plant species as daily diet and use of natural health products (NHPs) as medication urged the scientists to conduct extensive research on the antioxidant and AChE inhibitory effects of phenolic compounds in the treatment of AD (9).
The genus Leutea is represented by 11 taxa widespread throughout central Asia and Iran (10). Recently, this genus has been categorized as Ferula group (Ferula, Dorema, and Leutea) (11,12). Since Dorema and Ferula genera have strong antioxidant activity, this study was conducted on Leutea avicenniae for the first time (12, 13).
The extracts and diastereoisomers of Ferula lutea were examined for antioxidant activity and AChE inhibitory effects. The ethyl acetate extract had the strongest antioxidant activity (IC 50 = 12.8 ± 1.29 µg/mL) (13). Another investigation reported antioxidant and antihemolytic activities of Ferula gummosa Boiss. and its extract exerted moderate antioxidant activity (IC 50 = 579.6 ± 19.4 µg/mL) which was comparable with vitamin C (14).
In this study, we report the results of the antioxidant and AChE inhibitory effects of L. avicennia extract and it's identified compounds isolated from the methanol fraction.

Methods
Extraction and fractionation 700g of the air dried aerial parts of plant was macerated with 80% methanol (4 L ×5) at room temperature and was concentrated with an evaporator in low temperature (40 °C). The crude extract (160 g) was sequentially fractionated with petroleum ether, chloroform, ethyl acetate, and methanol (each 4 L) respectively, to obtain four fractions.

Acetylcholinestrase (AChE) inhibitory activity of Fractions
AChE inhibitory effect of petroleum ether, chloroform, ethyl acetate, and methanol fractions were carried out by the Ellman′s method (16). Galantamine was used as a positive control and distilled water as negative control.
In this method, 100 µL of 0.1 mM sodium phosphate buffer (pH = 8.0), 20 µL DTNB (5,5′-Dithiobis (2-nitrobenzoic acid) 20 µL of sample solution, 2 µL of AChE solution were added into 96 micro plates and were incubated for 15 min at 25 °C and then acetylcholine iodide (100 µL of 0.05 mM water solution) was added as substrate. Acetylcholinestrase activity was evaluated by measuring the absorbance at 412 nm by an ELISA plate reader for 3.0 min at 25 °C. The concentration of the compound which inhibited 50% of acetylcholinestrase activity (IC 50 ) was calculated (Table 1).

DPPH radical scavenging activity
The DPPH method is based on the free radical scavenging activity (17). briefly, 2 mL of DPPH solution in methanol (4×10 -5 g/mL) was prepared and added to 1 mL of different concentrations of sample solution in methanol (0.1, 0.5 and 1.0 mg/mL). The mixture was kept in the dark for 30 min at room temperature. The antioxidant activity was measured by Shimadzu, UV/VIS Isolated Compounds and Biological Activity of Leutea avicennia model 160A spectrophotometer at 517 nm.
All the measurements were done in triplicate. The IC 50 values were calculated as means ± SD and butylated hydroxytoluene (BHT) was used as positive control.

Acetylcholinestrase inhibitory and antioxidant activity of isolated compounds
AChE inhibitory and antioxidant effects of isolated compounds: quercetin, astragalin, caffeic acid, p-coumaric acid and salicylic acid were examined by the Ellman′s and DPPH methods respectively for determination of effective compounds (Table 1).

Results and Discussion
L. avicennia is an endemic plant of Iran and the purpose of this study was evaluation of antioxidant and AchE inhibitory effects of the plant extract, fractions, and isolated compounds. According to the antioxidant test, methanol fraction exerted strong free radical scavenging activity (IC 50 = 49.8 ± 7.0 µg/mL) Regarding the inhibition of acethylcholinestrase, methanol fraction showed inhibitory activity (205.10 ± 3.3 µg/mL) among other fraction and caffeic acid (IC 50 = 12.06 ± 2.01 µg/mL) was stronger than other isolated compounds. The antioxidant activity and AChE inhibitory assay of the total extract, fractions, and compounds are listed in the Table 1.
In previous study, quercetin (IC 50 =19.8 µg/mL). and 3-methoxy quercetin (IC 50 =37.9 µg/mL) isolated from Agrimonia had remarkable AChE inhibitory effect (16). Also, effect of quercetin on triple transgenic mice of Alzheimer's disease model has been studied and protective effects on performance, emotional and cognitive behavior have been confirmed on brain cells (30). In another study conducted in 2015, pharmacokinetics and mechanism of neuroprotective effect of quercetin was studied in mice representing strong protective effects of this compound on the brain cells (31).

Acetylcholinestrase inhibitory and antioxidant activity of isolated compounds
AChE inhibitory and antioxidant effects of isolated compounds: quercetin, astragalin, caffeic acid, p-coumaric acid and salicylic acid were examined by the Ellman's and DPPH methods respectively for determination of effective compounds (Table 1).

Acetylcholinestrase inhibitory and antioxidant activity of isolated compounds
AChE inhibitory and antioxidant effects of isolated compounds: quercetin, astragalin, caffeic acid, p-coumaric acid and salicylic acid were examined by the Ellman's and DPPH methods respectively for determination of effective compounds (Table 1).

Acetylcholinestrase inhibitory and antioxidant activity of isolated compounds
AChE inhibitory and antioxidant effects of isolated compounds: quercetin, astragalin, caffeic acid, p-coumaric acid and salicylic acid were examined by the Ellman's and DPPH methods respectively for determination of effective compounds (Table 1).

Conclusion
This study was conducted for the first time on Leutea avicennia, and showed this plant is rich of phenolic compounds that exhibited appropriate antioxidant and anti-acetylcholinesterase properties. Caffeic acid is one of the substances which showed potent activity in both assays, therefore methanol fraction of L.avicennia and their components probably could be used in treatment of neurodegenerative disease, so in-vivo animal model is worthwhile and recommended.